Fig. 6: GPNMB-mutation inhibited macrophage foaming.

A BMDMs from WT ApoE^−/−, HET ApoE^−/−, and Gpnmb^R150X ApoE^−/− mice were treated with OxLDL for 24 h, followed by BODIPY staining (left upper) or Dil-OxLDL for 12 h (left lower), and images were taken by immunofluorescence microscopy. N = 3 independent experiments. The quantitative analysis of total fluorescence intensity of BODIPY^TM (middle, n = 32/25/24) and Dil-OxLDL (right, n = 42/16/9). B The quantitative analysis of total fluorescence intensity of Dil-OxLDL in four groups of BMDMs from WT and Gpnmb^R150X mice treated with Dil-OxLDL for 12 h (mean ± SEM, N = 3 independent experiments, n = 3/group for each experiment). Representative FACS plots for the changes of GPNMB expression (C), the histogram of MFI of BODIPY in BMDMs from WT and Gpnmb^R150X mice treated with or without OxLDL for 48 h (D), and quantitative analysis of total fluorescence intensity of BODIPY in four groups of BMDMs in D (C, mean ± SEM, N = 4 independent experiments). E Representative images of WT BMDMs and Gpnmb^R150X BMDMs treated with Dil-OxLDL for 12 h (Red), followed by BODIPY^TM staining (Green) (left) and the localization of Dil-OxLDL with LDs quantified with Mandar overlap R-value analysis (right) (mean ± SEM, N = 3 independent experiments, n = 13 images each group). F Representative images of expression of SEC61β (Blue) in WT BMDMs and Gpnmb^R150X BMDMs treated with Dil-OxLDL for 12 h (Red) and stained with followed by BODIPY^TM staining (Green), N = 3 independent experiments. G Representative images of expression of MANNⅡ and KDEL (Red) in WT BMDMs and Gpnmb^R150X BMDMs treated with OxLDL for 24 h, followed by BODIPY^TM staining (Green), N = 3 independent experiments. H The localization of BODIPY^TM-labeled LDs (upper) or Dil-OxLDL (lower) with ER quantified with PCC (mean ± SEM, n = 8/4/5/4 in the upper figure, n = 5/5/9/10/4/4 in the lower figure). I Representative images of expression of RAB7, RAB11A, or SEC22B (Red) in WT BMDMs and Gpnmb^R150X BMDMs treated with OxLDL for 24 h, followed by BODIPY^TM staining (Green), N = 3 independent experiments. J The localization of BODIPY^TM-labeled LDs with Vesicles quantified with PCC (mean ± SEM, N = 3 independent experiments, n = 4/5/10/10/6/6/8/7/7/8). K Representative images of WT BMDMs and Gpnmb^R150X BMDMs treated with Dil-OxLDL for 12 h (Red) and stained with Mito-Tracker (Magenta), followed by BODIPY^TM staining (Green) (left) and the localization of BODIPY^TM-labeled LDs (middle, n = 51/44) or Dil-OxLDL (right, mean ± SEM, n = 9/10) with mitochondria quantified with Pearson’s Correlation Coefficient (PCC), N = 3 independent experiments. L Pathway enrichment analysis assessing GO terms for biological processes based on up-regulated proteins in Gpnmb^R150X BMDMs compared to WT BMDM, identified by mass spectrometry analysis. Selected pathways are shown, ranked by Counts. Differentially expressed proteins were determined with the Limma program at p-adj (adjusted p value) <0.05 and logFC (log fold change) > 1. M Sankey diagram of pathway enrichment analysis assessing GO terms for biological processes based on up-regulated proteins related to vesicle-trafficking in Gpnmb^R150X BMDMs compared to WT BMDM, identified by mass spectrometry analysis. Selected pathways are shown, ranked by Counts. Differentially expressed proteins were determined with the Limma program at p-adj (adjusted p value) <0.05 and logFC (log fold change) > 1. Data are expressed as mean ± SEM. P values were calculated by unpaired Student t-test (B, C, E, H, J, K) or one-way ANOVA (A). Differences are significant for *P  <  0.05, **P  <  0.01, ***P  <  0.001, ****P  <  0.0001.